title: The Legionella pneumophila GTPase activating protein LepB accelerates Rab1 deactivation by a non-canonical hydrolytic mechanism.
abstract: GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine.
authors.affiliation: Program in Molecular Medicine and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. Ashwini.Mishra@umassmed.edu
authors.forename: Ashwini K
authors.initials: AK
authors.lastname: Mishra
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Del Campo
authors.forename: Robert E
authors.initials: RE
authors.lastname: Collins
authors.forename: Craig R
authors.initials: CR
authors.lastname: Roy
authors.forename: David G
authors.initials: DG
authors.lastname: Lambright
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D016952:Legionella pneumophila / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D055672:Static Electricity; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D020690:GTPase-Activating Proteins; C482639:LepB protein, Legionella pneumophila; D014443:Tyrosine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020691:rab GTP-Binding Proteins
keywords: GAP; GTPase; GTPase Activating Protein; Host-Pathogen Interactions; Legionella pneumophila; LepB; Rab; Rab Proteins; Rab1; X-ray Crystallography